In the present article, we report on the kinetics of brain penetration in rats of the H3R antagonist 1,1'-[1,1'-biphenyl-4,4'-diylbis(methylene)]bis-[piperidine] (1), which had shown a favorable in vitro pharmacological profile and in vivo potency in preventing scopolamine-induced amnesia. Two different approaches were employed: high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) and ex vivo binding against the labeled agonist [(3)H]-(R)-α-methylhistamine ([(3)H]RAMHA). Starting from the structure of 1, the rigid piperidine ring was replaced by a flexible dipropylamino group (see 2) or by a morpholino ring (see 3), endowed with lower basicity. The effect of replacement on rat plasma and brain disposition in the 24 h after administration was analyzed. High (μM) and persistent concentrations of 1 were found in rat plasma, while plasma levels were significantly lower (range: 0-200 nM) for the other two derivatives. This could be explained, among other factors, by the higher stability, observed for 1, to liver metabolic cleavage. The applied chemical modulation had an important effect on in vivo brain disposition, as, despite the comparable physico-chemical properties, 2 did not show the tendency to accumulate within the brain, as stated by its brain vs. plasma concentration ratios, if compared to 1. These structureproperty relationships should be taken into account in the pharmacokinetic optimization of new series of H3 receptor antagonists.